The Effect of Protein Electrostatic Interactions on Globular Protein–Polymer Block Copolymer Self-Assembly

Mutation of a superfolder green fluorescent protein (GFP) was used to design GFP variants with formal net charges of 0, −8, and −21, providing a set of three proteins in which the total charge is varied to tune protein–protein interactions while controlling for the protein size and tertiary structure. After conjugating poly­(<i>N</i>-isopropylacrylamide) (PNIPAM) to each of these three GFP variants, the concentrated solution phase behavior of these three block copolymers is studied using a combination of small-angle X-ray scattering (SAXS), depolarized light scattering (DPLS), and turbidimetry to characterize their morphologies. The electrostatic repulsion between supercharged GFP suppresses ordering, increasing the order–disorder transition concentration (<i>C</i><sub>ODT</sub>) and decreasing the quality of the ordered nanostructures as measured by the full width at half-maximum of the primary scattering peak. By contrast, the charge distribution of the neutrally charged GFP results in its largest dipole moment, calculated about the protein’s center of mass, among the three GFP variants and a self-complementary Janus-like electrostatic surface potential that enhances nanostructure formation. The different electrostatic properties result in different protein–protein interactions that affect the high temperature morphologies, including the formation of macrophase separated or homogeneous micellar phases and the smaller hexagonal ordering window of the supercharged GFP. Small improvements in the quality of the ordered nanostructures of GFP(−21)-PNIPAM can be achieved through protein–divalent cation interactions. Therefore, varying protein charge and electrostatics is demonstrated as a method of tuning the magnitude and directionality of protein–protein interactions to control self-assembly.